Microbiology (2013), 159, 2626–2638 DOI 10.1099/mic.0.071019-0 Editor’s Choice Sulfite oxidation in the purple sulfur bacterium Allochromatium vinosum: identification of SoeABC as a major player and relevance of SoxYZ in the process Christiane Dahl, Bettina Franz,3 Daniela Hensen,4 Anne Kesselheim and Renate Zigann Correspondence Institut fu¨r Mikrobiologie & Biotechnologie, Rheinische Friedrich-Wilhelms-Universita¨t Bonn, Christiane Dahl Meckenheimer Allee 168, 53115 Bonn, Germany [email protected] In phototrophic sulfur bacteria, sulfite is a well-established intermediate during reduced sulfur compound oxidation. Sulfite is generated in the cytoplasm by the reverse-acting dissimilatory sulfite reductase DsrAB. Many purple sulfur bacteria can even use externally available sulfite as a photosynthetic electron donor. Nevertheless, the exact mode of sulfite oxidation in these organisms is a long-standing enigma. Indirect oxidation in the cytoplasm via adenosine-59- phosphosulfate (APS) catalysed by APS reductase and ATP sulfurylase is neither generally present nor essential. The inhibition of sulfite oxidation by tungstate in the model organism Allochromatium vinosum indicated the involvement of a molybdoenzyme, but homologues of the periplasmic molybdopterin-containing SorAB or SorT sulfite dehydrogenases are not encoded in genome-sequenced purple or green sulfur bacteria. However, genes for a membrane-bound polysulfide reductase-like iron–sulfur molybdoprotein (SoeABC) are universally present. The catalytic subunit of the protein is predicted to be oriented towards the cytoplasm. We compared the sulfide- and sulfite-oxidizing capabilities of A. vinosum WT with single mutants deficient in SoeABC or APS reductase and the respective double mutant, and were thus able to prove that SoeABC is the major sulfite-oxidizing enzyme in A. vinosum and probably also in other phototrophic sulfur bacteria. The genes also occur in a large number of chemotrophs, indicating a general importance of SoeABC for sulfite oxidation in the cytoplasm. Furthermore, we showed that the periplasmic sulfur substrate-binding protein SoxYZ is needed in parallel to the Received 1 July 2013 cytoplasmic enzymes for effective sulfite oxidation in A. vinosum and provided a model for the Accepted 11 September 2013 interplay between these systems despite their localization in different cellular compartments. INTRODUCTION purple sulfur bacterium that can also utilize thiosulfate and sulfite (Imhoff et al., 1998; Weissgerber et al., 2011). The Anoxygenic purple sulfur bacteria like the gammaproteo- first major process in the oxidation of thiosulfate and bacterium Allochromatium vinosum, a member of the sulfide is the formation of intracellularly stored sulfur family Chromatiaceae, are able to derive energy from light globules. The oxidation of thiosulfate to sulfate with sulfur by using reduced sulfur compounds as photosynthetic globules as an intermediate involves the periplasmic Sox electron donors. The substrates of purple sulfur bacteria proteins SoxYZ, SoxB, SoxXAK and SoxL (Pott & Dahl, primarily include sulfide and elemental sulfur (Imhoff, 1998; Hensen et al., 2006; Welte et al., 2009). For the 2005a). A. vinosum is a metabolically especially versatile oxidation of sulfide, A. vinosum possesses the genetic equipment for at least three different enzymes catalysing 3Present address: Institut fu¨r Medizinische Mikrobiologie und this reaction, namely the soluble periplasmic flavocyto- Krankenhaushygiene, Zentrum der Hygiene, Universita¨tsklinikum chrome c and the membrane-bound sulfide : quinone Frankfurt, Paul-Ehrlich-Straße 40, 60596 Frankfurt am Main, Germany. oxidoreductases SqrD and SqrF. The last two are both 4Present address: Merck Millipore, Boulevard Industrial Park, Padge predicted to be oriented towards the periplasm (Reinartz Road, Nottingham NG9 2JR, UK. et al., 1998; Gregersen et al., 2011; Weissgerber et al., 2011). Abbreviation: APS, adenosine-59-phosphosulfate. In A. vinosum, the sulfur globules are deposited in the Two supplementary tables are available with the online version of this same cellular compartment as the periplasmic thiosulfate- paper. and sulfide-oxidizing enzymes. This is evidenced by the 2626 071019 G 2013 SGM Printed in Great Britain Sulfite oxidation in A. vinosum presence of signal peptide-encoding sequences in the genes Recently, involvement of a putative heterotrimeric mem- for the three structural proteins constituting the sulfur brane-bound complex named SoeABC (sulfite-oxidizing globule envelope (Pattaragulwanit et al., 1998; Prange et al., enzyme) in sulfite oxidation in the cytoplasm of the 2004; Frigaard & Dahl, 2008). For further oxidation of organosulfonate-degrading chemotrophic alphaproteobac- stored sulfur, the so-called Dsr system is of essential terium Ruegeria pomeroyi has been reported, although not importance (Pott & Dahl, 1998; Dahl et al., 2005; Lu¨bbe rigorously documented, by Lehmann et al. (2012). The et al., 2006; Sander et al., 2006). The product of the Dsr SoeABC protein appears to be a member of the complex pathway is sulfite, generated by a reverse-acting dissimilat- iron–sulfur molybdoproteins and consists of an NrfD/PsrC- ory sulfite reductase (DsrAB), an enzyme that is well like membrane anchor (SoeC) and two cytoplasmic known to be located in the cytoplasm (Hipp et al., 1997; subunits: an iron–sulfur protein (SoeB) and a molybdopro- Pott & Dahl, 1998; Frigaard & Dahl, 2008). tein with an N-terminal iron–sulfur cluster binding site The last step is the oxidation of sulfite, yielding sulfate as (SoeA). It should be noted that SoeABC and the periplasmic the final product. Sulfate formation from sulfite is Sor-type sulfite dehydrogenases belong to completely energetically favourable and carried out by a wide range different families of molybdoenzymes. SorA and relatives of organisms (Simon & Kroneck, 2013). Many sulfite- belong to the sulfite oxidase proteins, which bind a single oxidizing enzymes are located outside the cytoplasmic molybdopterin without a second nucleotide. SoeA falls into membrane (in the periplasm in Gram-negative bacteria). the molybdopterin-binding MopB superfamily. In many The best-characterized enzyme belonging to this group, characterized members of this family molybdopterin is present SorAB, stems from Starkeya novella and consists of a in the form of a dinucleotide, with two molybdopterin dinu- molybdopyranopterin cofactor-carrying subunit (SorA) cleotide units per molybdenum. Similarities are obvious and a monohaem cytochrome c (SorB) (Kappler et al., within each molybdoenzyme family sequence, whereas no 2000; Kappler & Bailey, 2005). SorA-type molybdoproteins significant homologies can be detected between members of without a SorB subunit have been termed SorT (D’Errico different families (Kisker et al., 1998). Genes encoding et al., 2006; Wilson & Kappler, 2009), but recently this proteins related to SoeABC are present in purple as well as discrimination has been questioned (Simon & Kroneck, green sulfur bacteria, and in the past years have repeatedly 2013). A second option for oxidation of sulfite in the been speculated to be involved in the oxidation of sulfite periplasm is the Sox system. It has been shown that sulfite generated by the Dsr system in the cytoplasm (Frigaard & is accepted in vitro as a substrate of the reconstituted Sox Bryant, 2008; Frigaard & Dahl, 2008). Notably, soeABC-like system from the chemotroph Paracoccus denitrificans genes co-localize with dsr genes not only in the meta- (Friedrich et al., 2001). Notably, Friedrich and coworkers genome-derived sequence of another bacterium that belongs proved this reaction to be independent of the presence of to the Roseobacter clade like R. pomeroyi (Lenk et al., 2012), SoxCD, a molybdohaemoprotein catalysing the six-elec- but also in several green sulfur bacteria and in Halorhodospira tron oxidation of SoxY-cysteine-bound persulfide to halophila, a purple sulfur bacterium of the family Ecto- sulfone sulfur. Purple bacteria that form sulfur globules thiorhodospiraceae (Dahl, 2008; Frigaard & Dahl, 2008). during thiosulfate oxidation contain the Sox system, albeit Independent experimental evidence for the involvement of a without the SoxCD proteins (Hensen et al., 2006; Meyer molybdoprotein was obtained by inhibition of sulfite et al., 2007; Frigaard & Dahl, 2008). oxidation in A. vinosum with the molybdate-antagonist tungstate (Dahl, 1996). Oxidation of sulfite generated by the cytoplasmic Dsr proteins via the periplasmic pathways described would first In this work, we aimed to obtain a clearer picture of the require transport across the cytoplasmic membrane. proteins involved in the oxidation of sulfite in purple sulfur However, such transport is probably not necessary in bacteria and chose A. vinosum as the model organism. An organisms that contain a further well-characterized sulfite array of strains carrying single and multiple deletions of oxidation pathway: it is firmly established that a number of relevant genes was studied with regard to the oxidation of purple as well as green anoxygenic phototrophic sulfur externally supplied and internally generated sulfite. bacteria oxidize sulfite in the cytoplasm using an indirect Thereby, we identified AvSoeABC (Alvin_2491-2489) as a pathway via adenosine-59-phosphosulfate (APS) catalysed major player in the oxidation of sulfite. In addition, we by APS reductase and ATP sulfurylase (Dahl, 1996; Sa´nchez present evidence that SoxYZ is also essential in